Competition and Synergy of Arp2/3 and Formins in Nucleating Actin Waves
Preprint posted on 13 September 2023https://www.biorxiv.org/content/10.1101/2023.09.13.557508v1.full
Background．Actin dynamics play fundamental roles in various cell biological aspects, ranging from cell division, motility, as well as growth. A peculiar phenomenon of cycling rounds of actin polymerization and disassembly, referred asactin waves, is known to occur in many cell types and has been linked to several processes like migration and plasma membrane rearrangements (Inagaki and Katsuno, 2017). Actin polymerization is orchestrated by two main classes of nucleators, namelyForminsand theArp2/3complex, which respectively drive elongation of preexisting filaments and formation of new actin branches (Lappalainen et al. 2022). After being described and studied separately for many years, Formins and Arp2/3 are increasingly recognized to work in concert in many circumstances. Still, how their functions are coordinated represent an active area of investigation. In this preprint, the authors employ actin waves as a model tostudy the interplay between Formins and Arp2/3 in Mast cells．总的来说,他们发现previously unrecognized synergy between the two classes of nucleators, as well as a competition for the common upstream regulator Cdc42.
Key findings of this preprint
Formins precede Arp2/3 in coordinated actin waves
作者用超分辨率显微镜和various fluorescent reporters for the Formin mDia3 and FMNL1 and the Arp2/3 complex in order to catch a potential oscillatory and coordinated movement of these factors at the plasma membrane. Arp2/3 was recruited in all actin waves analyzed and in a punctate pattern, highlighting its importance and specialized function in this context. When present in the same cortical actin wave, Arp2/3 recruitment was consistently seen to lag behind Formins in the cycling steps of actin waves (Figure 1). The authors suggest that Formin-mediated actin polymerization could act as a trigger by providing preexisting filaments where Arp2/3 could be recruited to nucleate new branches.
|Figure 1 – Live imaging of Formin FMNL1 and Arp3 at cortical waves.|
Molecular mechanism of Formins recruitment to waves
To grasp the mechanism of Formin recruitment to the plasma membrane, the authors overexpressed various fluorescent Formin mutants and found that the GTPase binding domain, together with the releasement of the autoinhibited form of the protein are necessary to timely recruit Formins before Arp2/3 and in a rhythmic fashion (Figure 2).
|Figure 2 – Representative picture of kymographs showing actin waves of a constitutively active FMNL1 and a C-terminal FMNL1 portion together with Arp2/3. Releasement from autoinhibition (left) results in well-defined and rhythmic FMNL1 waves, whereas mutation in the GTPase binding domain (right) results in abrogation of a defined pattern of Formin wave.|
Requirements for actin nucleator recruitment to waves
Blocking actin dynamics by cytochalasin-D treatment allowed the authors to uncouple actin oscillation from membrane recruitment of actin-nucleator factors. Only the Formins and Arp2/3 upstream regulator Cdc42 was able to maintain membrane recruitment in this condition, whereas waves of Arp2/3, FMNL1 or mDia3 were all abolished, highlighting their need for interaction with actin.
Cdc42 competition rather than actin competition dictates waves coordination
封锁Arp2/3活动ck - 666治疗creased Formin and Cdc42 activity and recruitment to cortical waves. Arp2/3 recruitment dampened Formin-mediated actin polymerization, likely by competing for Cdc42 binding. Indeed, Cdc42 can bind both FMNL1 and WASp (the factor guiding Arp2/3 activity downstream of Cdc42) in a mutually exclusive way. Moreover, Arp2/3 activity was observed to somehow downregulate Cdc42 activity. The authors thus suggested that Arp2/3 recruitment functions as negative feedback to Formin at every cycle of an actin wave by competing for Cdc42 binding and activity.
Why I chose this preprint
Regulation of actin dynamics is a fascinating aspect in cell biology with lots of unanswered questions. The authors used very nice microscopy techniques to get further insights of this process. The evidence provided by the authors challenges the current simplistic view depicting competition between Formins and Arp2/3 for monomer actin and add another layer of complexity by showing Cdc42 as a critical regulator of actin nucleator activities.
Questions to the authors
- Would it be possible to generate fluorescent labelled knock-in to visualize formins and Arp2/3 endogenous localization and activities?
- How could Arp2/3 activity restrain Cdc42 function?
- What has been the most challenging part and what the most exciting part of your work for this story?
- Inagaki N., Katsuno H. “Actin Waves: Origin of Cell Polarization and Migration?” (2017),Trends in Cell Biology, 27 (7), pp. 515-526.
- Lappalainen, P., Kotila, T., Jégou, A. et al. “Biochemical and mechanical regulation of actin dynamics”. (2022), Nat Rev Mol Cell Biol 23, 836–852. https://doi.org/10.1038/s41580-022-00508-4
Posted on: 27 October 2023Read preprint
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